Substituted hexahydropyrrolo[1,2-a]pyrazines, octahydropyrido[1,2-a]-pyrazines and decahydropyrazino[1,2-a]azepines

ABSTRACT

Novel substituted hexahydropyrrolo[1,2-a]pyrazines, octahydropyrido[1,2-a]-pyrazines and decahydropyrazino[1,2-a]azepines, use of these compounds as pharmaceutical compositions, pharmaceutical compositions comprising the compounds, and a method of treatment employing these compounds and compositions. The compounds show a high and selective binding affinity to the histamine H3 receptor indicating histamine H3 receptor antagonistic, inverse agonistic or agonistic activity. As a result, the compounds are useful for the treatment of diseases and disorders related to the histamine H3 receptor.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/453,106 filed on Jun. 3, 2003, and claims the benefit of Danishapplication no. PA 2002 00863 filed on Jun. 6, 2002, and U.S.provisional application No. 60/387,047 filed on Jun. 7, 2002, thedisclosure of each of which is hereby incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates to novel substitutedhexahydropyrrolo[1,2-a]pyrazines, octahydropyrido[1,2-a]pyrazines anddecahydropyrazino[1,2-a]azepines, to the use of these compounds aspharmaceutical compositions, to pharmaceutical compositions comprisingthe compounds, and to a method of treatment employing these compoundsand compositions. The present compounds show a high and selectivebinding affinity to the histamine H3 receptor indicating histamine H3receptor antagonistic, inverse agonistic or agonistic activity. As aresult, the compounds are useful for the treatment of diseases anddisorders related to the histamine H3 receptor.

BACKGROUND OF THE INVENTION

The existence of the histamine H3 receptor has been known for severalyears and the receptor is of current interest for the development of newmedicaments. Recently, the human histamine H3 receptor has been cloned.The histamine H3 receptor is a presynaptic autoreceptor located both inthe central and the peripheral nervous system, the skin and in organssuch as the lung, the intestine, probably the spleen and thegastrointestinal tract. Recent evidence suggests that the H3 receptorshows intrinsic, constitutive activity, in vitro as well as in vivo (ieit is active in the absence of an agonist. Compounds acting as inverseagonists can inhibit this activity. The histamine H3 receptor has beendemonstrated to regulate the release of histamine and also of otherneurotransmitters such as serotonin and acetylcholine. A histamine H3receptor antagonist or inverse agonist would therefore be expected toincrease the release of these neurotransmitters in the brain. Ahistamine H3 receptor agonist, on the contrary, leads to an inhibitionof the biosynthesis of histamine and an inhibition of the release ofhistamine and also of other neurotransmitters such as serotonin andacetylcholine. These findings suggest that histamine H3 receptoragonists, inverse agonists and antagonists could be important mediatorsof neuronal activity. Accordingly, the histamine H3 receptor is animportant target for new therapeutics.

Several publications disclose the preparation and use of histamine H3agonists and antagonists. Most of these are imidazole derivatives.However, recently some imidazole-free ligands of the histamine H3receptor have been described (see eg Linney et al., J. Med. Chem. 2000,43, 2362-2370; U.S. Pat. No. 6,316,475, WO 01/66534, WO 01/74810.

In view of the art's interest in histamine H3 receptor agonists, inverseagonists and antagonists, novel compounds which interact with thehistamine H3 receptor would be a highly desirable contribution to theart. The present invention provides such a contribution to the art beingbased on the finding that a novel class of substitutedhexahydropyrrolo[1,2-a]pyrazines, octahydropyrido[1,2-a]pyrazines anddecahydropyrazino[1,2-a]azepines has a high and specific affinity to andpotency at the histamine H3 receptor.

Compounds having a certain similarity to the compounds of the presentinvention have previously been prepared, and their biological propertieshave been investigated, cf. Decosta et al., J. Med. Chem. 36 (16)2311-2320 (1993) and Bromidge et al., Bioorg. Med. Chem. Lett. 12 (10)1357-1360 (2002). However, these references do not disclose that thesecompounds may have a histamine H3 receptor antagonistic or agonisticactivity.

Due to their interaction with the histamine H3 receptor, the presentcompounds are useful in the treatment of a wide range of conditions anddisorders in which an interaction with the histamine H3 receptor isbeneficial. Thus, the compounds may find use e.g. in the treatment ofdiseases of the central nervous system, the peripheral nervous system,the cardiovascular system, the pulmonary system, the gastrointestinalsystem and the endocrinological system.

SUMMARY OF THE INVENTION

The invention relates to a compound of the general formula (I):

wherein

-   n is 1, 2 or 3,-   R^(1a) and R^(1b) independently are hydrogen, C₁₋₆-alkyl,    C₂₋₆-alkenyl, C₂₋₆-alkynyl, C₃₋₈-cycloalkyl, C₃₋₈-cycloalkenyl or    fluoro,-   R² is hydrogen, C₁₋₆-alkyl, C₂₋₆-alkenyl, C₂₋₆-alkynyl,    C₃₋₈-cycloalkyl or C₃₋₈-cycloalkenyl,-   X is    R^(3a), R^(3b), R^(3c) and R^(3d) independently are hydrogen,    halogen, C₁₋₆-alkyl or C₃₋₈-cycloalkyl, or R^(3a) and R^(3b), R^(3a)    and R^(3c) or R^(3c) and R^(3d) can be taken together to form a    C₁₋₆alkylene bridge,-   R⁴, R⁵, R⁶ and R⁷ independently are hydrogen, halogen, C₁₋₆alkyl or    C₃₋₈-cycloalkyl,-   Z is    R⁸, R⁹, R¹⁰, R¹¹ and R¹² independently are    -   hydrogen, cyano, nitro, halogen, carboxy, guanidino, or amidino,    -   C₁₋₆alkyl, C₁₋₆alkoxy, C₂₋₆-alkenyl, C₂₋₆-alkynyl,        C₁₋₆alkylsulfonyl, C₁₋₆alkylsulfinyl, C₁₋₆-alkylthio,        C₁₋₆-alkylcarbonyl, C₃₋₈-cycloalkyl, C₃₋₈-cycloalkylcarbonyl,        C₃₋₈-cycloalkenyl, aryl, arylsulfonyl, arylsulfinyl or arylthio,    -   each of which may optionally be substituted with one or more        groups selected from cyano, nitro, halogen, carboxy, guanidino,        amidino, trifluoromethyl, trifluoromethoxy, —NR¹³R⁴, —NHC(═O)R¹⁵        or —C(═O)NR¹³R¹⁴,    -   —NR³R⁴, —NHC(═O)R⁵, —OC(═O)NR³R¹⁴, —NHC(═O)OR¹⁶ or —C(═O)NR³R⁴,        or        R⁸ and R⁹, R⁹ and R¹⁰, R¹⁰ and R¹¹, or R¹¹ and R¹² can be taken        together to form a bridge selected from C₁₋₆-alkylene,        —O—(CH₂)_(n)—O— and —O—(CH₂)_(n)—,-   o is 1, 2, 3, 4 or 5,-   R¹³, R¹⁴ and R¹⁵ independently are hydrogen or-   aryl, C₁₋₆-alkyl, C₂₋₆-alkenyl, C₂₋₆-alkynyl, C₃₋₈-cycloalkyl or    C₃₋₈-cycloalkenyl,-   each of which may optionally be substituted with one or more groups    selected from cyano, nitro and halogen,-   R¹⁶ is-   aryl, C₁₋₆-alkyl, C₂₋₆-alkenyl, C₂₋₆-alkynyl, C₃₋₈-cycloalkyl or    C₃₋₈-cycloalkenyl,-   each of which may optionally be substituted with one or more groups    selected from cyano, nitro and halogen,-   as well as any diastereomer or enantiomer or tautomeric form thereof    including mixtures of these or a pharmaceutically acceptable salt    thereof.    Definitions

In the structural formulae given herein and throughout the presentspecification, the following terms have the indicated meaning:

The term “halogen” means F, Cl, Br or I.

The term “C₁₋₆-alkyl” as used herein represents a saturated, branched orstraight hydrocarbon group having from 1 to 6 carbon atoms. TypicalC₁₋₆-alkyl groups include, but are not limited to, methyl, ethyl,n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl,hexyl and the like.

The term “C₂₋₆-alkenyl” as used herein represents a branched or straighthydrocarbon group having from 3 to 8 carbon atoms and at least onedouble bond. Typical C₂₋₆-alkenyl groups include, but are not limitedto, ethenyl, 1-propenyl, 2-propenyl, isopropenyl, 1,3-butadienyl,1-butenyl, 2-butenyl, 1-pentenyl, 2-pentenyl, 1-hexenyl, 2-hexenyl,1-ethylprop-2-enyl, 1,1-(dimethyl)prop-2-enyl, 1-ethylbut-3-enyl,1,1-(dimethyl)but-2-enyl, and the like.

The term “C₂₋₆-alkynyl” as used herein represents a branched or straighthydrocarbon group having from 2 to 6 carbon atoms and at least onetriple bond. Typical C₂₋₆-alkynyl groups include, but are not limitedto, vinyl, 1-propynyl, 2-propynyl, isopropynyl, 1,3-buta-dynyl,1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 1-hexynyl, 2-hexynyl,1-ethylprop-2-ynyl, 1,1-(dimethyl)prop-2-ynyl, 1-ethylbut-3-ynyl,1,1-(dimethyl)but-2-ynyl, and the like.

The term “C₁₋₆-alkoxy” as used herein refers to the radical—O—C₁₋₆-alkyl, wherein C₁₋₆-alkyl is as defined above. Representativeexamples are methoxy, ethoxy, n-propoxy, isopropoxy, butoxy, sec-butoxy,tert-butoxy, pentoxy, isopentoxy, hexoxy, isohexoxy and the like.

The term “C₁₋₆-alkylthio as used herein refers to the radical—S—C₁₋₆-alkyl, wherein C₁₋₆-alkyl represents a saturated, branched orstraight hydrocarbon groups having from 1 to 6 carbon atoms as definedabove. Representative examples are methylthio, ethylthio, propylthio,butylthio, pentylthio, hexylthio, and the like

The term “C₁₋₆-alkylsulfonyl” as used herein refers to the radical—S(═O)₂—C₁₋₆-alkyl, wherein C₁₋₆-alkyl represents a saturated, branchedor straight hydrocarbon groups having from 1 to 6 carbon atoms asdefined above. Representative examples are methylsulfonyl,ethylsulfonyl, propylsulfonyl, butylsulfonyl, pentylsulfonyl,hexylsulfonyl, and the like.

The term “C₁₋₆-alkylsulfinyl” as used herein refers to the radical—S(═O)—C₁₋₆-alkyl, wherein C₁₋₆-alkyl represents a saturated, branchedor straight hydrocarbon groups having from 1 to 6 carbon atoms asdefined above. Representative examples are methylsulfinyl,ethylsulfinyl, propylsulfinyl, butylsulfinyl, pentylsulfinyl,hexylsulfinyl, and the like.

The term “C₃₋₈-cycloalkyl” as used herein represents a monocyclic,carbocyclic group having from from 3 to 8 carbon atoms. Representativeexamples are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclooctyl, and the like.

The term “C₃₋₈-cycloalkenyl” as used herein represents a monocyclic,carbocyclic, non-aromatic group having from 3 to 8 carbon atoms and atleast one double bond. Representative examples are cyclopropenyl,cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyland the like.

The term “aryl” as used herein is intended to include carbocyclicaromatic ring systems such as phenyl, biphenylyl, naphthyl, anthracenyl,phenanthrenyl, fluorenyl, indenyl, pentalenyl, azulenyl and the like.Aryl is also intended to include the partially hydrogenated derivativesof the carbocyclic systems enumerated above. Non-limiting examples ofsuch partially hydrogenated derivatives are 1,2,3,4-tetrahydronaphthyl,1,4-dihydronaphthyl and the like.

Certain of the above defined terms may occur more than once in thestructural formulae, and upon such occurrence each term shall be definedindependently of the other.

The term “optionally substituted” as used herein means that the groupsin question are either unsubstituted or substituted with one or more ofthe substituents specified. When the groups in question are substitutedwith more than one substituent the substituents may be the same ordifferent.

The term “treatment” as used herein means the management and care of apatient for the purpose of combating a disease, disorder or condition.The term is intended to include the delaying of the progression of thedisease, disorder or condition, the alleviation or relief of symptomsand complications, and/or the cure or elimination of the disease,disorder or condition. The patient to be treated is preferably a mammal,in particular a human being.

DESCRIPTION OF THE INVENTION

In one embodiment of the invention n is 1.

In another embodiment n is 2.

In yet another embodiment R^(1a) and R^(1b) are hydrogen.

In still another embodiment R² is hydrogen.

In a further embodiment X is

wherein R^(3a), R^(3b), R^(3c), R^(3d), R⁴, R⁵, R⁶ and R⁷ are as definedfor formula (I).

In yet a further embodiment X is —C(═O)—CH₂—CH₂—C(═O)—,—C(═O)—O—CH₂—CH₂— or

In still a further embodiment Z is

wherein R⁸ to R¹² are as defined for formula (I).

In one embodiment thereof R⁸ to R¹² independently are

-   -   hydrogen, cyano, nitro, halogen, carboxy, guanidino, or amidino,    -   C₁₋₆alkyl, C₁₋₆-alkoxy, C₂₋₆-alkenyl, C₂₋₆-alkynyl,        C₁₋₆-alkylsulfonyl, C₁₋₆-alkylsulfinyl, C₁₋₆-alkylthio,        C₁₋₆-alkylcarbonyl, C₃₋₈-cycloalkyl, C₃₋₈-cycloalkylcarbonyl,        C₃₋₈-cycloalkenyl, aryl, arylsulfonyl, arylsulfinyl or arylthio,    -   each of which may optionally be substituted with one or more        groups selected from cyano, nitro, halogen, carboxy, guanidino,        amidino, trifluoromethyl, trifluoromethoxy, —NR¹³R¹⁴,        —NHC(═O)R¹⁵ or —C(═O)NR¹³R¹⁴, and wherein aryl is selected from        phenyl, biphenylyl, naphthyl, anthracenyl, phenanthrenyl,        fluorenyl, indenyl, pentalenyl, azulenyl,        1,2,3,4-tetrahydronaphthyl, or 1,4-dihydronaphthyl,    -   —NR¹³R¹⁴, —NHC(═O)R⁵, —OC(═O)NR³R⁴, —NHC(═O)OR¹⁶ or        —C(═O)NR¹³R¹⁴, or        R⁸ and R⁹, R⁹ and R¹⁰, R¹⁰ and R¹¹, or R¹¹ and R¹² can be taken        together to form a bridge selected from C₁₋₆-alkylene,        —O—(CH₂)_(o)—O— and —O—(CH₂)_(o)—,

In yet a further embodiment Z is

wherein R¹⁰ to R¹¹ are as defined for formula (I).

In an embodiment thereof R¹⁰ and R” are independently hydrogen,C₁₋₆alkoxy, halogen or trifluoromethyl.

In another embodiment thereof at least one of R¹⁰ and R¹¹ is differentfrom hydrogen.

The compounds of the present invention may be chiral, and it is intendedthat any enantiomers, as separated, pure or partially purifiedenantiomers or racemic mixtures thereof are included within the scope ofthe invention.

Furthermore, when a double bond or a fully or partially saturated ringsystem or more than one center of asymmetry or a bond with restrictedrotatability is present in the molecule diastereomers may be formed. Itis intended that any diastereomers, as separated, pure or partiallypurified diastereomers or mixtures thereof are included within the scopeof the invention.

Furthermore, some of the compounds of the present invention may exist indifferent tautomeric forms and it is intended that any tautomeric forms,which the compounds are able to form, are included within the scope ofthe present invention.

The present invention also encompasses pharmaceutically acceptable saltsof the present compounds. Such salts include pharmaceutically acceptableacid addition salts, pharmaceutically acceptable metal salts, ammoniumand alkylated ammonium salts. Acid addition salts include salts ofinorganic acids as well as organic acids. Representative examples ofsuitable inorganic acids include hydrochloric, hydrobromic, hydroiodic,phosphoric, sulfuric, nitric acids and the like. Representative examplesof suitable organic acids include formic, acetic, trichloroacetic,trifluoroacetic, propionic, benzoic, cinnamic, citric, fumaric,glycolic, lactic, maleic, malic, malonic, mandelic, oxalic, picric,pyruvic, salicylic, succinic, methanesulfonic, ethanesulfonic, tartaric,ascorbic, pamoic, bismethylene salicylic, ethanedisulfonic, gluconic,citraconic, aspartic, stearic, palmitic, EDTA, glycolic, p-aminobenzoic,glutamic, benzenesulfonic, p-toluenesulfonic acids and the like. Furtherexamples of pharmaceutically acceptable inorganic or organic acidaddition salts include the pharmaceutically acceptable salts listed inJ. Pharm. Sci. 1977, 66, 2, which is incorporated herein by reference.Examples of metal salts include lithium, sodium, potassium, magnesiumsalts and the like. Examples of ammonium and alkylated ammonium saltsinclude ammonium, methylammonium, dimethylammonium, trimethylammonium,ethylammonium, hydroxyethylammonium, diethylammonium, butylammonium,tetramethylammonium salts and the like.

Also intended as pharmaceutically acceptable acid addition salts are thehydrates, which the present compounds are able to form.

The acid addition salts may be obtained as the direct products ofcompound synthesis. In the alternative, the free base may be dissolvedin a suitable solvent containing the appropriate acid, and the saltisolated by evaporating the solvent or otherwise separating the salt andsolvent.

The compounds of the present invention may form solvates with standardlow molecular weight solvents using methods well known to the personskilled in the art. Such solvates are also contemplated as being withinthe scope of the present invention.

The invention also encompasses prodrugs of the present compounds, whichon administration undergo chemical conversion by metabolic processesbefore becoming active pharmacological substances. In general, suchprodrugs will be functional derivatives of the present compounds, whichare readily convertible in vivo into the required compound of theformula (I). Conventional procedures for the selection and preparationof suitable prodrug derivatives are described, for example, in “Designof Prodrugs”, ed. H. Bundgaard, Elsevier, 1985.

The invention also encompasses active metabolites of the presentcompounds.

The compounds of the present invention interact with the histamine H3receptor and are accordingly useful for the treatment of a wide varietyof conditions and disorders in which histamine H3 receptor interactionsare beneficial.

Accordingly, in another aspect the present invention relates to acompound of the general formula (I) as well as any diastereomer orenantiomer or tautomeric form thereof including mixtures of these or apharmaceutically acceptable salt thereof for use as a pharmaceuticalcomposition.

The invention also relates to pharmaceutical compositions comprising, asan active ingredient, at least one compound of the formula (I) or anydiastereomer or enantiomer or tautomeric form thereof including mixturesof these or a pharmaceutically acceptable salt thereof together with oneor more pharmaceutically acceptable carriers or diluents.

Furthermore, the invention relates to the use of a compound of thegeneral formula (I) as well as any diastereomer or enantiomer ortautomeric form thereof including mixtures of these or apharmaceutically acceptable salt thereof for the preparation of apharmaceutical composition for the treatment of disorders and diseasesrelated to the histamine H3 receptor.

In still another aspect, the invention relates to a method for thetreatment of diseases and disorders related to the histamine H3 receptorthe method comprising administering to a subject in need thereof aneffective amount of a compound of the formula (I) or any diastereomer orenantiomer or tautomeric form thereof including mixtures of these or apharmaceutically acceptable salt thereof or a pharmaceutical compositioncomprising the same.

In one aspect the invention relates to compounds with histamine H3receptor antagonistic activity or inverse agonistic activity which mayaccordingly be useful in the treatment of a wide range of conditions anddisorders in which histamine H3 receptor blockade is beneficial.

In another aspect the invention relates to compounds with histamine H3receptor agonistic activity and which may accordingly be useful in thetreatment of a wide range of conditions and disorders in which histamineH3 receptor activation is beneficial.

In a preferred embodiment of the invention the present compounds areused for the preparation of a pharmaceutical composition for thereduction of weight.

In a preferred embodiment of the invention the present compounds areused for the preparation of a pharmaceutical composition for thetreatment of overweight or obesity.

In another preferred embodiment of the invention the present compoundsare used for the preparation of a pharmaceutical composition for thesuppression of appetite or satiety induction.

In a further preferred embodiment of the invention the present compoundsare used for the preparation of a pharmaceutical composition for theprevention and/or treatment of disorders and diseases related tooverweight or obesity such as atherosclerosis, hypertension, IGT(impaired glucose tolerance), diabetes, especially type 2 diabetes(NIDDM (non-insulin dependent diabetes mellitus)), dyslipidaemia,coronary heart disease, gallbladder disease, osteoarthritis and varioustypes of cancer such as endometrial, breast, prostate and colon cancers.

In yet a further preferred embodiment of the invention the presentcompounds are used for the preparation of a pharmaceutical compositionfor the prevention and/or treatment of eating disorders such as bulimiaand binge eating.

In a further preferred embodiment of the invention the present compoundsare used for the preparation of a pharmaceutical composition for thetreatment of IGT.

In a further preferred embodiment of the invention the present compoundsare used for the preparation of a pharmaceutical composition for thetreatment of type 2 diabetes. Such treatment includes inter aliatreatment for the purpose of delaying or prevention of the progressionfrom IGT to type 2 diabetes as well as delaying or prevention of theprogression from non-insulin requiring type 2 diabetes to insulinrequiring type 2 diabetes.

The compounds of the present invention may also be used for thetreatment of airway disorders such as asthma, as anti-diarrhoeals andfor the modulation of gastric acid secretion.

Furthermore, the compounds of the present invention may be used for thetreatment of diseases associated with the regulation of sleep andwakefulness and for the treatment of narcolepsy and attention deficitdisorder.

Moreover, the compounds of the invention may be used as CNS stimulantsor as sedatives.

The present compounds may also be used for the treatment of conditionsassociated with epilepsy. Additionally, the present compounds may beused for the treatment of motion sickness and vertigo. Furthermore, theymay be useful as regulators of hypothalamo-hypophyseal secretion,antidepressants, modulators of cerebral circulation, and in thetreatment of irritable bowel syndrome.

Further, the compounds of the present invention may be used for thetreatment of dementia and Alzheimer's disease.

The compounds of the present invention may also be useful for thetreatment of allergic rhinitis, ulcer or anorexia.

The compounds of the present invention may furthermore be useful for thetreatment of migraine, see McLeod et al., The Journal of Pharmacologyand Experimental Therapeutics 287 (1998), 43-50, and for the treatmentof myocardial infarction, see Mackins et al., Expert Opinion onInvestigational Drugs 9 (2000), 2537-2542.

In a further aspect of the invention treatment of a patient with thepresent compounds is combined with diet and/or exercise.

In a further aspect of the invention the present compounds areadministered in combination with one or more further active substancesin any suitable ratio(s). Such further active agents may be selectedfrom antiobesity agents, antidiabetics, antidyslipidemic agents,antihypertensive agents, agents for the treatment of complicationsresulting from or associated with diabetes and agents for the treatmentof complications and disorders resulting from or associated withobesity.

Thus, in a further aspect of the invention the present compounds areadministered in combination with one or more antiobesity agents orappetite regulating agents.

Such agents may be selected from the group consisting of CART (cocaineamphetamine regulated transcript) agonists, NPY (neuropeptide Y)antagonists, MC4 (melanocortin 4) agonists, MC3 (melanocortin 3)agonists, orexin antagonists, TNF (tumor necrosis factor) agonists, CRF(corticotropin releasing factor) agonists, CRF BP (corticotropinreleasing factor binding protein) antagonists, urocortin agonists, β3adrenergic agonists such as CL-316243, AJ-9677, GW-0604, LY362884,LY377267 or AZ-40140, MSH (melanocyte-stimulating hormone) agonists, MCH(melanocyte-concentrating hormone) antagonists, CCK (cholecystokinin)agonists, serotonin re-uptake inhibitors such as fluoxetine, seroxat orcitalopram, serotonin and noradrenaline re-uptake inhibitors, mixedserotonin and noradrenergic compounds, 5HT (serotonin) agonists,bombesin agonists, galanin antagonists, growth hormone, growth factorssuch as prolactin or placental lactogen, growth hormone releasingcompounds, TRH (thyreotropin releasing hormone) agonists, UCP 2 or 3(uncoupling protein 2 or 3) modulators, leptin agonists, DA agonists(bromocriptin, doprexin), lipase/amylase inhibitors, PPAR (peroxisomeproliferator-activated receptor) modulators, RXR (retinoid X receptor)modulators, TR β agonists, AGRP (Agouti related protein) inhibitors,opioid antagonists (such as naltrexone), exendin-4, GLP-1 and ciliaryneurotrophic factor.

In one embodiment of the invention the antiobesity agent is leptin.

In another embodiment the antiobesity agent is dexamphetamine oramphetamine.

In another embodiment the antiobesity agent is fenfluramine ordexfenfluramine.

In still another embodiment the antiobesity agent is sibutramine.

In a further embodiment the antiobesity agent is orlistat.

In another embodiment the antiobesity agent is mazindol or phentermine.

In still another embodiment the antiobesity agent is phendimetrazine,diethylpropion, fluoxetine, bupropion, topiramate or ecopipam.

In yet another embodiment the antiobesity agent is growth hormone, agrowth factor such as prolactin or placental lactogen, or a growthhormone releasing compound.

In yet a further aspect the present compounds are administered incombination with one or more antidiabetic agents.

Relevant antidiabetic agents include insulin, insulin analogues andderivatives such as those disclosed in EP 0 792 290 (Novo Nordisk A/S),eg N^(εB29)-tetradecanoyl des (B30) human insulin, EP 0 214 826 and EP 0705 275 (Novo Nordisk A/S), eg Asp^(B28) human insulin, U.S. Pat. No.5,504,188 (Eli Lilly), eg Lys^(B28) Pro^(B29) human insulin, EP 0 368187 (Aventis), eg Lantus®, which are all incorporated herein byreference, GLP-1 derivatives such as those disclosed in WO 98/08871(Novo Nordisk A/S), which is incorporated herein by reference, as wellas orally active hypoglycaemic agents.

The orally active hypoglycaemic agents preferably comprise imidazolines,sulfonylureas, biguanides, meglitinides, oxadiazolidinediones,thiazolidinediones, insulin sensitizers, α-glucosidase inhibitors,agents acting on the ATP-dependent potassium channel of the β-cells egpotassium channel openers such as those disclosed in WO 97/26265, WO99/03861 and WO 00/37474 (Novo Nordisk A/S) which are incorporatedherein by reference, or mitiglinide, or a potassium channel blocker,such as BTS-67582, nateglinide, glucagon antagonists such as thosedisclosed in WO 99/01423 and WO 00/39088 (Novo Nordisk A/S and AgouronPharmaceuticals, Inc.), which are incorporated herein by reference,GLP-1 agonists such as those disclosed in WO 00/42026 (Novo Nordisk A/Sand Agouron Pharmaceuticals, Inc.), which are incorporated herein byreference, DPP-IV (dipeptidyl peptidase-IV) inhibitors, PTPase (proteintyrosine phosphatase) inhibitors, inhibitors of hepatic enzymes involvedin stimulation of gluconeogenesis and/or glycogenolysis, glucose uptakemodulators, GSK-3 (glycogen synthase kinase-3) inhibitors, compoundsmodifying the lipid metabolism such as antilipidemic agents, compoundslowering food intake, PPAR (peroxisome proliferator-activated receptor)and RXR (retinoid X receptor) agonists, such as ALRT-268, LG-1268 orLG-1069.

In one embodiment of the invention the present compounds areadministered in combination with insulin or an insulin analogue orderivative, such as N^(εB29)-tetradecanoyl des (B30) human insulin,Asp^(B28) human insulin, Lys^(B28) Pro^(B29) human insulin, Lantus®, ora mix-preparation comprising one or more of these.

In a further embodiment of the invention the present compounds areadministered in combination with a sulfonylurea eg tolbutamide,chlorpropamide, tolazamide, glibenclamide, glipizide, glimepiride,gliclazide or glyburide.

In another embodiment of the invention the present compounds areadministered in combination with a biguanide eg metformin.

In yet another embodiment of the invention the present compounds areadministered in combination with a meglitinide eg repaglinide ornateglinide.

In still another embodiment of the invention the present compounds areadministered in combination with a thiazolidinedione insulin sensitizereg troglitazone, ciglitazone, pioglitazone, rosiglitazone, isaglitazone,darglitazone, englitazone, CS-011/C₁₋₁₀₃₇ or T 174 or the compoundsdisclosed in WO 97/41097, WO 97/41119, WO 97/41120, WO 00/41121 and WO98/45292 (Dr. Reddy's Research Foundation), which are incorporatedherein by reference.

In still another embodiment of the invention the present compounds maybe administered in combination with an insulin sensitizer eg such as GI262570, YM-440, MCC-555, JTT-501, AR-H039242, KRP-297, GW-409544,CRE-16336, AR-H049020, LY510929, MBX-102, CLX-0940, GW-501516 or thecompounds disclosed in WO 99/19313, WO 00/50414, WO 00/63191, WO00/63192, WO 00/63193 (Dr. Reddy's Research Foundation) and WO 00/23425,WO 00/23415, WO 00/23451, WO 00/23445, WO 00/23417, WO 00/23416, WO00/63153, WO 00/63196, WO 00/63209, WO 00/63190 and WO 00/63189 (NovoNordisk A/S), which are incorporated herein by reference.

In a further embodiment of the invention the present compounds areadministered in combination with an α-glucosidase inhibitor egvoglibose, emiglitate, miglitol or acarbose.

In another embodiment of the invention the present compounds areadministered in combination with an agent acting on the ATP-dependentpotassium channel of the β-cells eg tolbutamide, glibenclamide,glipizide, gliclazide, BTS-67582 or repaglinide.

In yet another embodiment of the invention the present compounds may beadministered in combination with nateglinide.

In still another embodiment, the present compounds are administered incombination with an antihyperlipidemic agent or antilipidemic agent, egcholestyramine, colestipol, clofibrate, gemfibrozil, lovastatin,pravastatin, simvastatin, probucol or dextrothyroxine.

In still another embodiment of the invention the present compounds areadministered in combination with an antilipidemic agent egcholestyramine, colestipol, clofibrate, gemfibrozil, lovastatin,pravastatin, simvastatin, probucol or dextrothyroxine.

In another aspect of the invention, the present compounds areadministered in combination with more than one of the above-mentionedcompounds eg in combination with metformin and a sulfonylurea such asglyburide; a sulfonylurea and acarbose; nateglinide and metformin;acarbose and metformin; a sulfonylurea, metformin and troglitazone;insulin and a sulfonylurea; insulin and metformin; insulin, metforminand a sulfonylurea; insulin and troglitazone; insulin and lovastatin;etc.

Furthermore, the present compounds may be administered in combinationwith one or more antihypertensive agents. Examples of antihypertensiveagents are β-blockers such as alprenolol, atenolol, timolol, pindolol,propranolol and metoprolol, ACE (angiotensin converting enzyme)inhibitors such as benazepril, captopril, enalapril, fosinopril,lisinopril, quinapril and ramipril, calcium channel blockers such asnifedipine, felodipine, nicardipine, isradipine, nimodipine, diltiazemand verapamil, and α-blockers such as doxazosin, urapidil, prazosin andterazosin. Further reference can be made to Remington: The Science andPractice of Pharmacy, 19^(th) Edition, Gennaro, Ed., Mack PublishingCo., Easton, Pa., 1995.

It should be understood that any suitable combination of the compoundsaccording to the invention with diet and/or exercise, one or more of theabove-mentioned compounds and optionally one or more other activesubstances are considered to be within the scope of the presentinvention.

Pharmaceutical Compositions

The compounds of the invention may be administered alone or incombination with pharmaceutically acceptable carriers or excipients, ineither single or multiple doses. The pharmaceutical compositionsaccording to the invention may be formulated with pharmaceuticallyacceptable carriers or diluents as well as any other known adjuvants andexcipients in accordance with conventional techniques such as thosedisclosed in Remington: The Science and Practice of Pharmacy, 19^(th)Edition, Gennaro, Ed., Mack Publishing Co., Easton, Pa., 1995.

The pharmaceutical compositions may be specifically formulated foradministration by any suitable route such as the oral, rectal, nasal,pulmonary, topical (including buccal and sublingual), transdermal,intracisternal, intraperitoneal, vaginal and parenteral (includingsubcutaneous, intramuscular, intrathecal, intravenous and intradermal)route, the oral route being preferred. It will be appreciated that thepreferred route will depend on the general condition and age of thesubject to be treated, the nature of the condition to be treated and theactive ingredient chosen.

Pharmaceutical compositions for oral administration include solid dosageforms such as capsules, tablets, dragees, pills, lozenges, powders andgranules. Where appropriate, they can be prepared with coatings such asenteric coatings or they can be formulated so as to provide controlledrelease of the active ingredient such as sustained or prolonged releaseaccording to methods well known in the art.

Liquid dosage forms for oral administration include solutions,emulsions, suspensions, syrups and elixirs.

Pharmaceutical compositions for parenteral administration includesterile aqueous and non-aqueous injectable solutions, dispersions,suspensions or emulsions as well as sterile powders to be reconstitutedin sterile injectable solutions or dispersions prior to use. Depotinjectable formulations are also contemplated as being within the scopeof the present invention.

Other suitable administration forms include suppositories, sprays,ointments, cremes, gels, inhalants, dermal patches, implants etc.

A typical oral dosage is in the range of from about 0.001 to about 100mg/kg body weight per day, preferably from about 0.01 to about 50 mg/kgbody weight per day, and more preferred from about 0.05 to about 10mg/kg body weight per day administered in one or more dosages such as 1to 3 dosages. The exact dosage will depend upon the frequency and modeof administration, the sex, age, weight and general condition of thesubject treated, the nature and severity of the condition treated andany concomitant diseases to be treated and other factors evident tothose skilled in the art.

The formulations may conveniently be presented in unit dosage form bymethods known to those skilled in the art. A typical unit dosage formfor oral administration one or more times per day such as 1 to 3 timesper day may contain of from 0.05 to about 1000 mg, preferably from about0.1 to about 500 mg, and more preferred from about 0.5 mg to about 200mg.

For parenteral routes, such as intravenous, intrathecal, intramuscularand similar administration, typically doses are in the order of abouthalf the dose employed for oral administration.

The compounds of this invention are generally utilized as the freesubstance or as a pharmaceutically acceptable salt thereof. One exampleis an acid addition salt of a compound having the utility of a freebase. When a compound of the formula (I) contains a free base such saltsare prepared in a conventional manner by treating a solution orsuspension of a free base of the formula (I) with a chemical equivalentof a pharmaceutically acceptable acid, for example, inorganic andorganic acids. Representative examples are mentioned above.Physiologically acceptable salts of a compound with a hydroxy groupinclude the anion of said compound in combination with a suitable cationsuch as sodium or ammonium ion.

For parenteral administration, solutions of the novel compounds of theformula (I) in sterile aqueous solution, aqueous propylene glycol orsesame or peanut oil may be employed. Such aqueous solutions should besuitable buffered if necessary and the liquid diluent first renderedisotonic with sufficient saline or glucose. The aqueous solutions areparticularly suitable for intravenous, intramuscular, subcutaneous andintraperitoneal administration. The sterile aqueous media employed areall readily available by standard techniques known to those skilled inthe art.

Suitable pharmaceutical carriers include inert solid diluents orfillers, sterile aqueous solution and various organic solvents. Examplesof solid carriers are lactose, terra alba, sucrose, cyclodextrin, talc,gelatine, agar, pectin, acacia, magnesium stearate, stearic acid orlower alkyl ethers of cellulose. Examples of liquid carriers are syrup,peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines,polyoxyethylene or water. Similarly, the carrier or diluent may includeany sustained release material known in the art, such as glycerylmonostearate or glyceryl distearate, alone or mixed with a wax. Thepharmaceutical compositions formed by combining the novel compounds ofthe formula (I) and the pharmaceutically acceptable carriers are thenreadily administered in a variety of dosage forms suitable for thedisclosed routes of administration. The formulations may conveniently bepresented in unit dosage form by methods known in the art of pharmacy.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules or tablets, eachcontaining a predetermined amount of the active ingredient, and whichmay include a suitable excipient. These formulations may be in the formof powder or granules, as a solution or suspension in an aqueous ornon-aqueous liquid, or as an oil-in-water or water-in-oil liquidemulsion.

If a solid carrier is used for oral administration, the preparation maybe tabletted, placed in a hard gelatine capsule in powder or pellet formor it can be in the form of a troche or lozenge. The amount of solidcarrier will vary widely but will usually be from about 25 mg to about 1g. If a liquid carrier is used, the preparation may be in the form of asyrup, emulsion, soft gelatine capsule or sterile injectable liquid suchas an aqueous or non-aqueous liquid suspension or solution.

A typical tablet, which may be prepared by conventional tablettingtechniques, may contain: Core: Active compound (as free compound or saltthereof) 5.0 mg Lactosum Ph. Eur. 67.8 mg Cellulose, microcryst.(Avicel) 31.4 mg Amberlite ® IRP88* 1.0 mg Magnesii stearas Ph. Eur.q.s. Coating: Hydroxypropyl methylcellulose approx. 9 mg Mywacett 9-40T** approx. 0.9 mg*Polacrillin potassium NF, tablet disintegrant, Rohm and Haas.**Acylated monoglyceride used as plasticizer for film coating.

If desired, the pharmaceutical composition of the invention may comprisethe compound of the formula (I) in combination with furtherpharmacologically active substances such as those described in theforegoing.

EXAMPLES

HPLC (Method A)

The reverse phase analysis was performed using UV detections at 214 and254 nm on a 218TP54 4.6 mm×150 mm C-18 silica column, which was elutedat 1 ml/min at 42° C. The column was equilibrated with 5% acetonitrile,85% water and 10% of a solution of 0.5% trifluoroacetic acid in waterand eluted by a linear gradient from 5% acetonitrile, 85% water and 10%of a solution of 0.5% trifluoroacetic acid to 90% acetonitrile and 10%of a solution of 0.5% trifluoroacetic acid over 15 min.

HPLC (Method B)

The reverse phase analysis was performed using a Alliance Waters 2695system fitted with a Waters 2487 dual band detector. UV detections werecollected using a Symmetry C18, 3.5 μm, 3.0 mm×100 mm column. The columnwas eluted with a linear gradient of 5-90% acetonitrile, 90-0% water,and 5% trifluoroacetic acid (1.0%) in water over 8 min at a flow-rate of1.0 min/min.

General Procedure (A)

A compound of formula (Iz) according to the invention may be preparedaccording to general procedure (A) as illustrated below:

-   -   wherein R^(1a), R^(1b), R^(3a), R^(3b), R^(3c), R^(3d), R², n,        and Z are as defined for formula (I)        Step A:

An amino acid of formula (II), which is protected at the amino groupwith a suitable protecting group (PG) known to a person skilled in theart and described in the literature (eg Protective Groups in OrganicSynthesis, Greene, T. W., Wuts, P. G. M., 2^(nd) edition, John Wiley &Sons, New York) is reacted with an amino acid ester or a suitable saltthereof under amide-coupling conditions known to a person skilled in theart, eg in the presence of a suitable coupling reagent such as asuitable carbodiimide alone or a suitable carbodiimide in combinationwith a suitable helping agent such as 1-hydroxybenzotriazole,1-hydroxy-7-azabenzotriazole, or3-hydroxy-3H-dihydrobenzo[d][1,2,3]triazin-4-one, optionally in thepresence of a base such as diisopropylethylamine or triethylamine togive an amide of formula (III), wherein R is the residue of an arbitraryalcohol or phenol.

Step B:

The protecting group of the amine is removed by a method known in theliterature (eg Protective Groups in Organic Synthesis, Greene, T. W.,Wuts, P. G. M., 2^(nd) edition, John Wiley & Sons, New York) to give theamine of formula (IV).

Step C:

The cyclization yielding a compound of formula (V) may be performed inthe presence of a base such as eg triethylamine or diisopropylamine at asuitable temperature.

Step D:

A reduction with a suitable reduction agent, such as eg lithiumaluminium hydride results in the formation of the bicyclic compound offormula (VI).

Step E:

A coupling with a suitable acid (VII) in the presence of a suitablecoupling reagent known to a person skilled in the art such as eg asuitable carbodiimide alone or in combination with a suitable helpingagent such as 1-hydroxybenzotriazole, 1-hydroxy-7-azabenzotriazole, or3-hydroxy-3H-dihydrobenzo[d][1,2,3]triazin-4-one, optionally in thepresence of a base such as diisopropylethylamine or triethylamineprovides a compound of formula (Iz).

General Procedure (B)

A compound of formula (Iy) according to the invention may be preparedaccording to general procedure (B) as illustrated below:

-   -   wherein R^(1a), R^(1b), R², R³, R^(3b), R^(3c), R^(3d), n and Z        are as defined for formula (I).        Step A:

An alcohol of the general formula (VIII) is reacted with a bi-activatedcarbonic acid equivalent known to a person skilled in the art eg4-nitrophenyl chloroformate, carbonyl diimidazole or triphosgene, togive an activated carbon ester equivalent (IX).

Step B:

The compound of formula (IX) is reacted with the amine (VI) at asuitable temperature in the presence of a base such asdiisopropylethylamine or triethylamine to yield a compound of formula(Iy).

General Procedure (C)

A compound of the general formula (1×) may be prepared according togeneral procedure (C) illustrated below:

-   -   wherein R^(1a), R^(1b), R², R⁴, R⁵, R⁶, R⁷, n and Z are as        defined for formula (I).        Step A:

The compound of formula (VI) is reacted with a fluoride of formula (X)in the presence of a suitable base such as triethylamine,diisopropylethylamine, or tert-buyltetra-mehylguanidine in a suitablesolvent such as dimethylsulfoxide, N,N-dimethylformamide,N-methylpyrrolidinone, tetrahydrofuran or dichloromethane at a suitabletemperature to provide the compound of formula (1×).

Example 1 General Procedure (A)1-(3,4-Dimethoxyphenyl)-4-((S)-hexahydropyrrolo[1,2-a]pyrazin-2-yl)butane-1,4-dione

Step A:(S)-2-(((Methoxycarbonyl)methyl)carbamoyl)pyrrolidine-1-carboxylic acidtert-butyl ester

At 0° C., 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride(8.82 g, 46 mmol) was added to a solution of 1-hydroxybenzotriazole(6.21 g, 46 mmol) and BOC-protected proline (10.0 g, 46 mmol) in amixture of N,N-dimethylformamide (60 ml) and dichloromethane (60 ml).The reaction mixture was stirred for 30 min at 0° C. Glycine methylester hydrochloride (5.77 g, 46 mmol) and ethyldiisopropylamine (24 ml)were added successively. The reaction mixture was stirred for 16 hours,while it was warming up to room temperature. It was diluted with ethylacetate (300 ml) and washed with a 10% aqueous solution of sodiumhydrogen sulphate (300 ml). The aqueous phase was extracted with ethylacetate (2×200 ml). The combined organic layers were washed with asaturated aqueous solution of sodium hydrogen carbonate (200 ml) anddried over magnesium sulphate. The solvent was removed in vacuo. Thecrude product was purified by flash chromatography on silica (200 g),using ethyl acetate as eluent, to give 10.25 g of(S)-2-(((methoxycarbonyl)methyl)carbamoyl)pyrrolidine-1-carboxylic acidtert-butyl ester.

¹H NMR (CDCl₃): δ1.50 (s, 9H); 1.80-2.40 (m, 4H); 3.20-3.60 (m, 2H);3.80 (s, 3H); 3.90-4.45 (m, 3H); 6.55 (br, 1H).

Step B: [((S)-Pyrrolidine-2-carbonyl)amino]acetic acid methyl estertrifluoroacetic acid salt

TFA

Trifluoroacetic acid (50 ml) was added to a solution of(S)-2-(((methoxycarbonyl)-methyl)carbamoyl)pyrrolidine-1-carboxylic acidtert-butyl ester (10.25 g, 36 mmol) in dichloromethane (50 ml). Thereaction mixture was stirred for 1.25 hours. The solvent was removed invacuo. The residue was dissolved in dichloromethane (50 ml). The solventwas removed in vacuo. The latter procedure was repeated once, to give15.4 g of the crude trifluoroacetic acid salt of[((S)-pyrrolidine-2-carbonyl)amino]acetic acid methyl ester, which wasused in the next step without purification.

¹H NMR (CDCl₃): δ2.05 (m, 3H); 2.50 (m, 1H); 3.50 (m, 2H); 3.75 (s, 3H);4.05 (ABX, 2H); 4.75 (m, 1H); 7.80 (t, 1H); 10.60 (br, 2H).

Step C: (S)-Hexahydropyrrolo[1,2-a]pyrazine-1,4-dione

Triethylamine (75 ml, 540 mmol) was added to a solution of the crudetrifluoroacetic acid salt of [((S)-pyrrolidine-2-carbonyl)amino]aceticacid methyl ester (15.4 g), which was isolated in the previous step, inmethanol (300 ml). The reaction mixture was heated to reflux for 16hours. It was cooled to room temperature. The solvent was removed invacuo. The residue was suspended in 2-propanol (150 ml). Theprecipitation was isolated and dried in vacuo to give 4.38 g of(S)-hexahydropyrrolo[1,2-a]pyrazine-1,4-dione.

¹H NMR (DMSO-d₆): δ1.85 (m, 3H); 2.15 (m, 1H); 3.35 (m, 2H); 3.55 (dd,1H); 4.00 (d, 1H); 4.15 (m, 1H); 8.05 (br, 1H).Step D: (S)-Octahydropyrrolo[1,2-a]pyrazine dihydrochloride salt

(S)-Hexahydropyrrolo[1,2-a]pyrazine-1,4-dione (3.08 g, 20 mmol) wasadded to a 1.0 M solution of lithium aluminium hydride intetrahydrofuran (100 ml, 100 mmol). The mixture was heated to reflux for1.5 hours. It was cooled to 0° C. Water (5 ml) was added carefullydropwise. A 1N solution of sodium hydroxide in water (5 ml) was addedcarfully dropwise. Water (10 ml) was added. The reaction mixture wasleft for 16 hours. The precipitation was removed by filtration through aplug of celite. A 2.8 M solution of hydrogen chloride in ethyl acetate(60 ml) was added to the filtrate. The solvent was removed in vacuo. Theresidue was dissolved in ethyl acetate. The solvent was removed invacuo, to give 5.0 g of the crude dihydrochloride salt of(S)-octahydropyrrolo[1,2-a]pyrazine, which was used for the next stepwithout further purification.

¹H NMR (DMSO-d₆): δ1.70-2.35 (m, 4H); 3.00-4.15 (m, 9H).

Step E:

At 0° C., 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride(479 mg, 2.5 mmol) was added to a solution of3-(3,4-dimethoxybenzoyl)propionic acid (596 mg, 2.5 mmol) and3-hydroxy-3H-dihydrobenzo[d][1,2,3]triazin-4-one (408 mg, 2.5 mmol) in amixture of dichloromethane (10 ml) and N,N-dimethylformamide (10 ml).The reaction mixture was stirred for 25 min at 0° C. A solution of thecrude dihydrochloride salt of (S)-octahydro-pyrrolo[1,2-a]pyrazine inN,N-dimethylformamide (4 ml) and ethyldiisopropylamine (3.0 ml, 17.5mmol) were added successively. The reaction mixture was stirred for 16hours, while it was warming up to room temperature. It was diluted withethyl acetate (200 ml) and washed with a saturated aqueous solution ofsodium hydrogen carbonate. The aqueous phase was extracted with ethylacetate (2×100 ml). The combined organic layers were dried overmagnesium sulphate. The solvent was removed in vacuo. The crude productwas purified by HPLC on a C18-reversed phase column, using a mixture of1% trifluoroacetic acid in water and acetonitrile as eluent, to give 148mg of the title compound.

¹H NMR (CDCl₃, 2 sets of signals): δ 1.45 (m, 1H); 1.60-2.10 (m, 4H);2.15 and 2.45 (both m, together 3H) 2.85 (m, 3H); 3.10 (m, 2H); 2.25 and4.05 (m and d, together 3H); 3.91 (s, 3H); 3.93 (s, 3H); 4.60 and 4.75(both d, together 1H); 6.90 (d, 1H); 7.60 (s, 1H); 7.70 (d, 1H); HPLC(method A): elution at 6.27 min; MS: Calc. for [M+H]⁺: 347; Found: 347.

The title compound was transferred into its hydrochloride salt, bydissolving it in ethyl acetate (5 ml). A 2.8 M solution of hydrogenchloride in ethyl acetate (2 ml) was added. The solvent was removed invacuo. The residue was dissolved in methanol (20 ml) and ethyl acetate(20 ml). The solvent was removed in vacuo.

C₁₉H₂₆N₂O₄.HCl.H₂O (346.43·3.46·18.02):

Calc.: C 56.92H 7.29 N 6.99; Found: C 56.91H 6.89 N 6.77.

Example 2 General Procedure (A)1-(4-Chlorophenyl)-4-((S)-hexahydropyrrolo[1,2-a]pyrazin-2-yl)butane-1,4-dione

279 mg of the title compound were prepared as described for1-(3,4-dimethoxy-phenyl)-4-((S)-hexahydropyrrolo[1,2-a]pyrazin-2-yl)butane-1,4-dione,using 3-(4-chlorobenzoyl) propionic acid instead of3-(3,4-dimethoxybenzoyl)propionic acid.

¹H NMR (CDCl₃, 2 sets of signals): δ 1.45 (m, 1H); 1.70-2.05 and 2.15(both m, together 6H); 2.40 and 2.70-3.00 (both m, together 3H);3.00-3.20 and 3.20-3.40 (both m, together 5H); 3.93 and 4.05 (both d,together 1H); 4.60 and 4.70 (both d, together 1H); 7.40 (d, 2H); 7.95(d, 2H); HPLC (method A): elution at 7.66 min; MS: Calc. for [M+H]⁺:321; Found: 321.

The title compound was transferred into its hydrochloride salt, bydissolving it in ethyl acetate (5 ml). A 2.8 M solution of hydrogenchloride in ethyl acetate (2 ml) was added. The solvent was removed invacuo. The residue was dissolved in methanol (20 ml) and ethyl acetate(20 ml). The solvent was removed in vacuo.

Example 3 General Procedure (A)1-(4-Chlorophenyl)-4-(octahydropyrido[1,2-a]pyrazin-2-yl)butane-1,4-dione

463 mg of the title compound were prepared as described for1-(3,4-dimethoxyphenyl)-4-((S)-hexahydropyrrolo[1,2-a]pyrazin-2-yl)butane-1,4-dione,using 1-(tert-butoxycarbonyl)piperidine 2 carboxylic acid instead ofBOC-protected proline and 3-(4-chlorobenzoyl)propanoic acid instead of3-(3,4-dimethoxybenzoyl)propionic acid.

¹H NMR (CDCl₃, 2 sets of signals): δ1.10-2.50 (m, 9H); 2.60-3.00 (m,5H); 3.20-3.40 (m, 3H); 3.75 and 3.90 (both d, together 1H); 4.40 and4.52 (both d, together 1H); 7.40 (d, 2H); 8.00 (d, 2H); HPLC (method A):elution at 7.79 min; MS: Calc. for [M+H]⁺: 335; Found: 335.

The title compound was transferred into its hydrochloride salt, bydissolving it in ethyl acetate (5 ml). A 2.8 M solution of hydrogenchloride in ethyl acetate (2 ml) was added. The solvent was removed invacuo. The residue was dissolved in methanol (20 ml) and ethyl acetate(20 ml). The solvent was removed in vacuo.

C₁₈H₂₃ClN₂O₂.HCl.½H₂O (334.85·36.46·½ 18.02):

Calc.: C 56.85H 6.63 N 7.37; Found: C 56.96H 6.57 N 7.35.

Example 4 General Procedure (A)1-(3-Fluoro-4-methoxyphenyl)-4-(octahydropyrido[1,2-a]pyrazin-2-yl)butane-1,4-dione

12 mg of the title compound were prepared as described for1-(3,4-dimethoxyphenyl)-4-((S)-hexahydropyrrolo[1,2-a]pyrazin-2-yl)butane-1,4-dione,using 1-(tert-butoxycarbonyl)piperidine 2 carboxylic acid instead ofBOC-protected proline and 3-(4-methoxy-3-fluorobenzoyl)propanoic acidinstead of 3-(3,4-dimethoxybenzoyl)propionic acid.

¹H NMR (CDCl₃, 2 sets of signals): δ1.25 (m, 2H); 1.55-2.50 (m, 8H);2.85 (m, 5H); 3.30 (m, 2H); 3.75 and 3.90 (dt and d, together 1H); 3.98(s, 3H); 4.40 and 4.55 (dt, and d, together 1H); 7.00 (t, 1H); 7.75 (d,1H); 7.85 (d, 1H); HPLC (method A): elution at 7.23 min; MS: Calc. for[M+H]⁺: 349; Found: 349.

The title compound was transferred into its hydrochloride salt, bydissolving it in ethyl acetate (5 ml). A 2.8 M solution of hydrogenchloride in ethyl acetate (2 ml) was added. The solvent was removed invacuo. The residue was dissolved in methanol (20 ml) and ethyl acetate(20 ml). The solvent was removed in vacuo.

Example 5 General Procedure (A)1-(3,4-Dimethoxyphenyl)-4-(octahydropyrido[1,2-a]pyrazin-2-yl)butane-1,4-dione

360 mg of the title compound were prepared as described for1-(3,4-dimethoxyphenyl)-4-((S)-hexahydropyrrolo[1,2-a]pyrazin-2-yl)butane-1,4-dione,using 1-(tert-butoxycarbonyl)piperidine 2 carboxylic acid instead ofBOC-protected proline.

¹H NMR (CDCl₃, 2 sets of signals): δ1.10-1.95, 2.00-2.30, and 2.40 (m,m, and t, together 10H); 2.65-2.95 (m, 4H); 3.20-3.45 (m, 3H); 3.75 and3.90 (d and m, together 1H); 3.95 (s, 3H); 3.97 (s, 3H); 4.40 and 4.45(both d, together 1H); 6.90 (d, 1H); 7.55 (s, 1H); 7.70 (d, 1H); HPLC(method A): elution at 6.52 min; MS: Calc. for [M+H]⁺: 361; Found: 361.

The title compound was transferred into its hydrochloride salt, bydissolving it in ethyl acetate (5 ml). A 2.8 M solution of hydrogenchloride in ethyl acetate (2 ml) was added. The solvent was removed invacuo. The residue was dissolved in methanol (20 ml) and ethyl acetate(20 ml). The solvent was removed in vacuo.

C₂₀H₂₈N₂O₄.HCl (360.46-36.46):

Calc.: C 60.52H 7.36 N 7.06; Found: C 58.90H 7.12 N 6.82.

Example 6 General Procedure (A)1-(3-Fluoro-4-methoxyphenyl)-4-((S)-octahydropyrido[1,2-a]pyrazin-2-yl)butane-1,4-dione

380 mg of the title compound were prepared as described for1-(3,4-dimethoxyphenyl)-4-((S)-hexahydropyrrolo[1,2-a]pyrazin-2-yl)butane-1,4-dione,using (S)-1-(tert-butoxycarbonyl)piperidine 2 carboxylic acid instead ofBOC-protected proline and 3-(3-fluoro-4-methoxybenzoyl)propionic acidinstead of 3-(3,4-dimethoxybenzoyl)propionic acid.

¹H NMR (CDCl₃, 2 sets of signals): δ1.25 (m, 2H); 1.50-2.45 (m, 8H);2.85 (m, 5H); 3.20-3.45 (d, 2H); 3.75 and 3.90 (both m, together 1H);3.95 (s, 3H); 4.40 and 4.55 (both m, together 1H); 7.00 (t, 1H); 7.75(d, 1H); 7.85 (d, 1H); HPLC (method A): elution at 7.27 min; MS: Calc.for [M+H]⁺: 349; Found: 349.

The title compound was transferred into its hydrochloride salt, bydissolving it in ethyl acetate (5 ml). A 2.8 M solution of hydrogenchloride in ethyl acetate (2 ml) was added. The solvent was removed invacuo. The residue was dissolved in methanol (20 ml) and ethyl acetate(20 ml). The solvent was removed in vacuo.

C₁₉H₂₅N₂O₃.HCl.⅓H₂O (390.89):

Calc.: C 58.38H 6.88 N 7.17; Found: C 58.37H 6.92 N 7.04.

Example 7 General Procedure (A)1-(3-Fluoro-4-methoxyphenyl)-4-((R)-octahydropyrido[1,2-a]pyrazin-2-yl)butane-1,4-dione

93 mg of the title compound were prepared as described for1-(3,4-dimethoxyphenyl)-4-((S)-hexahydropyrrolo[1,2-a]pyrazin-2-yl)butane-1,4-dione,using (R)-1-(tert-butoxycarbonyl)piperidine 2 carboxylic acid instead ofBOC-protected proline and 3-(3-fluoro-4-methoxybenzoyl)propionic acidinstead of 3-(3,4-dimethoxybenzoyl)propionic acid.

¹H NMR (CDCl₃, 2 sets of signals): δ1.25 (m, 2H); 1.50-2.45 (m, 8H);2.85 (m, 5H); 3.20-3.45 (d, 2H); 3.75 and 3.90 (both m, together 1H);3.95 (s, 3H); 4.40 and 4.55 (both m, together 1H); 7.00 (t, 1H); 7.75(d, 1H); 7.85 (d, 1H); HPLC (method A): elution at 7.20 min; MS: Calc.for [M+H]⁺: 349; Found: 349.

The title compound was transferred into its hydrochloride salt, bydissolving it in ethyl acetate (5 ml). A 2.8 M solution of hydrogenchloride in ethyl acetate (2 ml) was added. The solvent was removed invacuo. The residue was dissolved in methanol (20 ml) and ethyl acetate(20 ml). The solvent was removed in vacuo.

C₁₉H₂₅N₂O₃.HCl. 1/3H₂O (390.89):

Calc.: C 58.38H 6.88 N 7.17; Found: C 58.37H 6.92 N 7.04.

Example 8 General Procedure (B)(S)-Octahydropyrido[1,2-a]pyrazine-2-carboxylic acid2-(4-methoxyphenyl)ethyl ester

Preparation of (S)-Octahydropyrido[1,2-a]pyrazine (VI)

The dihydrochloride salt of (S)-octahydropyrido[1,2-a]pyrazine wasprepared as described in steps A to D of example 1 using(S)-1-(tert-butoxycarbonyl)piperidine carboxylic acid instead ofBOC-protected proline.

¹H NMR (DMSO-d₆): δ1.1.40-2.00 (m, 6H); 2.90-3.60 (m, 9H); 10.20 (br,2H); 12.15 (br, 1H).

Step A:

4-Nitrophenyl chloroformate (103 mg, 0.51 mmol) was added to a solutionof 2-(4-methoxyphenyl)ethanol (78 mg, 0.51 mmol) and pyridine (0.082 ml,1.02 mmol) in dichloromethane (15 ml). The reaction mixture was stirredat room temperature for 2.5 hours. It was diluted with dichloromethane(30 ml) and washed with water (3×30 ml). The organic layer was driedover magnesium sulphate.

Step B:

The solvent was removed in vacuo. The residue was dissolved inacetonitrile and added to a suspension of the dihydrochloride salt of(S)-octahydropyrido[1,2-a]pyrazine (138 mg, 0.64 mmol) and triethylamine(0.62 ml, 4.48 mmol) in dichloromethane (5 ml). The reaction mixture wasstirred for 16 hours at room temperature. It was diluted with ethylacetate (70 ml) and washed with a saturated aqueous solution of sodiumhydrogencarbonate (50 ml). The aqueous solution was extracted with ethylacetate (2×20 ml). The combined organic layers were dried over magnesiumsulphate. The solvent was removed in vacuo. The crude product waspurified by flash chromatography on silica (30 g), usingdichloromethane/methanol/25% aqueous ammonia (100:10:1) as eluent. Theresiude was dissolved in ethyl acetate (30 ml). It was extracted with a10% aqueous solution of sodium hydrogen-sulphate (20 ml). The aqueoussolution was made basic with a 1N sodium hydroxide solution and wasextracted with ethyl acetate (2×20 ml). The combined extracts were driedover magnesium sulphate. The solvent was removed in vacuo to give 27 mgof the title compound.

¹H NMR (CDCl₃): δ1.10-1.40 (m, 2H); 1.50-1.70 (m, 3H); 1.70-1.90 (m,2H); 1.95-2.20 (m, 2H); 2.50-3.10 (m, 4H); 2.90 (t, 2H); 3.80 (s, 3H);3.85-4.15 (m, 2H); 4.25 (t, 2H); 6.85 (d 2H); 7.12 (d, 2H); HPLC (methodA): elution at 7.80 min; MS: Calc. for [M+H]⁺: 319; Found: 319.

The title compound was transferred into its hydrochloride salt, bydissolving it in ethyl acetate (5 ml). A 2.8 M solution of hydrogenchloride in ethyl acetate (2 ml) was added. The solvent was removed invacuo. The residue was dissolved in methanol (20 ml) and ethyl acetate(20 ml). The solvent was removed in vacuo.

Example 9 General Procedure (B)(R)-Octahydropyrido[1,2-a]pyrazine-2-carboxylic acid2-(3-(trifluoromethyl)phenyl)ethyl ester

48 mg of the title compound was prepared as described for(S)-octahydropyrido[1,2-a]pyrazine-2-carboxylic acid2-(4-methoxyphenyl)ethyl ester, using2-(3-(trifluoromethyl)-phenyl)ethanol instead of2-(4-methoxyphenyl)ethanol.

¹H NMR (CDCl₃, 2 sets of signals): δ1.05-1.40 (m, 2H); 1.40-1.70 (m,3H); 1.70-1.95 (m, 2H); 1.95-2.25 (m, 2H); 2.55 (m, 1H); 2.70 (m, 1H);2.85 (m, 1H); 3.00 (m, 3H); 3.75, 3.95, and 4.05 (all m, together 2H);4.30 (m, 2H); 7.45 (m, 2H); 7.50 (m, 2H); HPLC method A: elution at 9.09min; MS: Calc. for [M+H]⁺: 357; Found: 357.

The title compound was transferred into its hydrochloride salt, bydissolving it in ethyl acetate (5 ml). A 2.8 M solution of hydrogenchloride in ethyl acetate (2 ml) was added. The solvent was removed invacuo. The residue was dissolved in methanol (20 ml) and ethyl acetate(20 ml). The solvent was removed in vacuo.

Example 10 General Procedure (C)[3,5-Difluoro-4-((R)-octahydropyrido[1,2-a]pyrazin-2-yl)phenyl]-[phenyl]methanone

(R)-Octahydropyrido[1,2-a]pyrazine

The dihydrochloride salt of (R)-octahydropyrido[1,2-a]pyrazine wassynthesized analogously to the dihydrochloride salt of(S)-octahydropyrrolo[1,2-a]pyrazine, using(R)-1-(tert-butoxycarbonyl)piperidine 2 carboxylic acid instead ofBOC-protected proline.

¹H NMR (DMSO-d₆): δ1.35-1.95 (m, 6H); 2.85-3.60 (m, 9H); 10.25, 10.45,and 12.25 (all br, together 3H).

Step A:

A mixture of the dihydrochloride salt of(R)-octahydropyrido[1,2-a]pyrazine (300 mg, 1.41 mmol), triethylamine(1.17 ml, 8.46 mmol) and 3,4,5-trifluorobenzophenone (332 mg, 1.41.mmol; commercially available eg at Interchim, France) indimethylsulfoxide (5 ml) was heated to 120° C. for 16 hours. Thereaction mixture was cooled to room temperature and diluted with asolution of potassium carbonate (2.0 g) in water (50 ml). It wasextracted with ethyl acetate (30 ml). The organic layer was dried overmagnesium sulphate. The solvent was removed in vacuo. The crude productwas purified by flash chromatography on silica (40 g), using first ethylacetate/heptane (1:1,500 ml) and afterwardsdichloromethane/methanol/−25% aqueous ammonia (100:10:1) as eluent, togive 132 mg of the title compound.

¹H NMR (CDCl₃, 2 sets of signals): δ 1.25 (m, 2H); 1.55 (d, 1H); 1.65(m, 2H); 1.80 (d, 1H); 2.10 (m, 2H); 2.45 (td, 1H); 2.80 (d, 1H); 2.90(d, 1H); 3.05 (t, 1H); 3.25 (d, 1H); 3.45 (m, 1H); 3.50 (t, 1H); 7.35(m, 2H); 7.50 (m, 2H); 7.60 (t, 1H); 7.75 (d, 2H); HPLC method B:elution at 4.25 min; MS: calc. for [M+H]⁺: 357; found: 357.

The title compound was transferred into its hydrochloride salt, bydissolving it in ethyl acetate (5 ml). A 2.8 M solution of hydrogenchloride in ethyl acetate (2 ml) was added. The solvent was removed invacuo. The residue was dissolved in methanol (20 ml) and ethyl acetate(20 ml). The solvent was removed in vacuo.

Example 11 General Procedure (C)(4-Fluorophenyl)-[4-((R)-octahydropyrido[1,2-a]pyrazin-2-yl)phenyl]methanone

340 mg of the title compound were prepared as described for[3,5-difluoro-4-((R)-octahydropyrido[1,2-a]pyrazin-2-yl)phenyl]-[phenyl]methanone,using 4,4′-difluorobenzophenone instead of 3,4,5-trifluorobenzophenone.

¹H NMR (CDCl₃, 2 sets of signals): δ 1.35 (m, 2H); 1.65 (m, 3H); 1.80(m, 1H); 2.05 (m, 2H); 2.35 (m, 1H); 2.65 (m, 1H); 3.40 (m, 2H); 3.10(m, 1H); 3.65 (d, 1H); 3.80 (d, 1H); 6.90 (d, 2H); 7.15 (t, 2H); 7.75(m, 4H); HPLC method A: elution at 8.46 min; MS: Calc. for [M+H]⁺: 339;Found: 339.

The title compound was transferred into its hydrochloride salt, bydissolving it in ethyl acetate (5 ml). A 2.8 M solution of hydrogenchloride in ethyl acetate (2 ml) was added. The solvent was removed invacuo. The residue was dissolved in methanol (20 ml) and ethyl acetate(20 ml). The solvent was removed in vacuo.

Example 12 General Procedure (C){[4-((R)-Octahydropyrido[1,2-a]pyrazin-2-yl)phenyl]}-{phenyl}methanone

180 mg of the title compound were prepared as described for[3,5-difluoro-4-((R)-octahydropyrido[1,2-a]pyrazin-2-yl)phenyl]-[phenyl]methanone,using 4-fluorobenzophenone instead of 3,4,5-trifluorobenzophenone.

¹H NMR (CDCl₃, 2 sets of signals): δ1.30 (m, 2H); 1.65 (m, 3H); 1.80 (m,1H); 2.05 (m, 2H); 2.35 (dt, 2H); 2.65 (m, 1H); 2.90 (m, 2H); 3.10 (dt,1H); 3.65 (d, 1H); 3.80 (d, 1H); 6.90 (d, 2H); 7.45 (m, 2H); 7.55 (m,1H); 7.75 (m, 2H); 7.80 (d, 2H); HPLC method A: elution at 8.12 min; MS:Calc. for [M+H]⁺: 321; Found: 321.

The title compound was transferred into its hydrochloride salt, bydissolving it in ethyl acetate (5 ml). A 2.8 M solution of hydrogenchloride in ethyl acetate (2 ml) was added. The solvent was removed invacuo. The residue was dissolved in methanol (20 ml) and ethyl acetate(20 ml). The solvent was removed in vacuo.

Example 13 General Procedure (#)(R)-2-[6-(4-Trifluoromethyl-phenyl)-pyridazin-3-yl]-octahydro-pyrido[1,2-a]pyrazine

A mixture of the dihydrochloride salt of(R)-octahydropyrido[1,2-a]pyrazine (649 mg, 3.05 mmol),diisopropylethylamine (1.17 ml, 6.72 mmol),3-chloro-6-(4-trifluoromethylphenyl)-pyridazine (290 mg, 1.12 mmol) anddimethylsulfoxide (1 ml) was heated in a closed vessel at 160° C. for 40min. The reaction mixture was cooled to room temperature, diluted withdichloromethane (10 ml) and washed with water (2×15 ml) and brine (15ml). The organic layer was dried over magnesium sulphate. The solventwas removed in vacuo to give 398 mg of the title compound as the freebase.

¹H NMR (400 MHz, CDCl₃) δ 1.34 (broad t, 2H), 1.69 (m, 3H), 1.83 (m,1H), 2.00-2.14 (m, 2H), 2.34 (dt, 1H), 2.81 (dd, 1H), 2.90 (broad d,2H), 3.25 (dt, 1H), 4.32 (d, J=11.6 Hz, 2H, 6.99 (d, J=9.6 Hz, 1H), 7.67(d, J=9.6 Hz, 1H), 7.72 (d, J=8.1 Hz, 2H), 8.12 (d, J=8.1 Hz, 2H).

HPLC-MS: m/z 362.8 (MH⁺); Rt: 2.66 min.

Pharmacological Methods

The ability of the compounds to interact with the histamine H3 receptorcan be determined by the following in vitro binding assays.

Binding assay I

The H3-receptor agonist ligand R-α-methyl[³H]histamine (RAMHA) isincubated with isolated rat cortex cell-membranes at 25° C. for 1 hour,followed by a filtration of the incubate through Whatman GF/B filters.Radioactivity retained on the filters is measured using a beta counter.

Male Wistar rats (150-200 g) are decapitated and cerebral cortex isquickly dissected out and frozen immediately on dry ice. Tissue is keptat −80° C. until membrane preparation. During the membrane preparationthe tissue is kept on ice all the time. Rat cerebral cortex ishomogenized in 10 volumes (w/w) ice-cold Hepes buffer (20 mM Hepes, 5 mMMgCl₂ pH 7.1 (KOH)+1 mg/ml bacitracin) using an Ultra-Turrax homogenizerfor 30 seconds. The homogenate is centrifuged at 140 g in 10 min. Thesupernatant is transferred to a new test tube and centrifuged for 30 minat 30 000 g. Pellet is resuspended in 5-10 ml Hepes buffer, homogenizedand centrifuged for 10 min at 30 000 g. This short centrifugation stepis repeated twice. After the last centrifugation the pellet isresuspended in 2-4 ml Hepes buffer and the protein concentration isdetermined. The membranes are diluted to a protein concentration of 5mg/ml using Hepes buffer, aliquoted and stored at −80° C. until use.

50 μl test-compound, 100 μl membrane (200 μg/ml), 300 μl Hepes bufferand 50 μl R-α-methyl[³H]histamine (1 nM) are mixed in a test tube. Thecompounds to be tested are dissolved in DMSO and further diluted in H₂Oto the desired concentrations. Radioligand and membranes are diluted inHepes buffer+1 mg/ml bacitracin. The mixture is incubated for 60 min at25° C. Incubation is terminated by adding 5 ml ice-cold 0.9% NaCl,followed by rapid filtration through Whatman GF/B filters pre-treatedfor 1 hour with 0.5% polyethyleneimine. The filters are washed with 2×5ml ice-cold NaCl. To each filter a 3 ml scintillation cocktail is addedand the radioactivity retained is measured with a Packard Tri-Carb betacounter.

IC₅₀ values are calculated by non-linear regression analysis of bindingcurves (6 points minimum) using the windows program GraphPad Prism,GraphPad software, USA.

Binding Assay II

The human H3 receptor is cloned by PCR and subcloned into the pcDNA3expression vector. Cells stably expressing the H3 receptor are generatedby transfecting the H3-expression vectors into HEK 293 cells and usingG418 to select for H3 clones. The human H3-HEK 293 clones are culturedin DMEM (GIBCO-BRL) with glutamax, 10% foetal calf serum, 1%penicillin/streptavidin and 1 mg/ml G 418 at 37° C. and 5% CO₂. Beforeharvesting, the confluent cells are rinsed with PBS and incubated withVersene (proteinase, GIBCO-BRL) for approximately 5 min. The cells areflushed with PBS and DMEM and the cell suspension collected in a tubeand centrifuged for 5-10 min at 420 g in a Heraeus Sepatech Megafuge1.0. The pellet is resuspended in 10-20 vol. Hepes buffer (20 mM Hepes,5 mM MgCl₂, pH 7.1 (KOH)) and homogenized for 10-20 seconds using anUltra-Turrax homogenizer. The homogenate is centrifuged for 30 min at 30000 g. The pellet is resuspended in 5-10 ml Hepes buffer, homogenized5-10 seconds with the Ultra-Turrax and centrifuged for 10 min at 30 000g. Following this centrifugation step, the membrane pellet isresuspended in 2-4 ml Hepes buffer, homogenized with a syringe or Teflonhomogenizer, and the protein concentration determined. The membranes arediluted to a protein concentration of 1-5 mg/ml in Hepes buffer,aliquoted and kept at −80° C. until use.

Aliquots of the membrane suspension are incubated for 60 min at 25° C.with 30 pM [¹²⁵I]-iodoproxifan, a known compound with high affinity forthe H3 receptor, and the test compound at various concentrations. Theincubation is stopped by dilution with ice-cold medium, followed byrapid filtration through Whatman GF/B filters pretreated for 1 hour with0.5% polyethyleneimine. The radioactivity retained on the filters iscounted using a Cobra II auto gamma counter. The radioactivity of thefilters is indirectly proportional to the binding affinity of the testedcompound. The results are analysed by nonlinear regression analysis.

When tested, the present compounds of the formula (I) generally show ahigh binding affinity to the histamine H3 receptor.

Functional Assay I

The ability of the compounds to interact with the histamine H3 receptoras agonists, inverse agonists and/or antagonists, is determined by an invitro functional assay utilizing membranes from HEK 293 cell expressingthe human H3 receptors.

The H3 receptor is cloned by PCR and subcloned into the pcDNA3expression vector. Cells stably expressing the H3 receptor are generatedby transfecting the H3-expression vectors into HEK 293 cells and usingG418 to select for H3 clones. The human H3-HEK 293 clones are culturedin DMEM with glutamax, 10% foetal calf serum, 1% penicillin/streptavidinand 1 mg/ml G418 at 37° C. and 5% CO₂.

The H3 receptor expressing cells are washed once with phosphate bufferedsaline (PBS) and harvested using versene (GIBCO-BRL). PBS is added andthe cells are centrifuged for 5 min at 364 g. The cell pellet isresuspended in stimulation buffer to a concentration of 1×10⁶ cells/ml.cAMP accumulation is measured using the Flash Plate® cAMP assay (NEN™Life Science Products). The assay is generally performed as described bythe manufacturer. Briefly, 50 μl cell suspension is added to each wellof the Flashplate which also contained 25 μl 40 μM isoprenaline, tostimulate cAMP generation, and 25 μl of test compound (either agonistsor inverse agonists alone, or agonist and antagonist in combination).The assay can be run in “agonist-mode” which means that the testcompound is added, in increasing concentration, on its own, to thecells, and cAMP is measured. If cAMP goes up, it is an inverse agonist;if CAMP does not change, it is a neutral antagonist, and if CAMP goesdown, it is an agonist. The assay can also be run in the“antagonist-mode” which means that a test compound is added, inincreasing concentrations, together with increasing concentrations of aknown H3 agonist (eg RAMHA). If the compound is an antagonist,increasing concentrations of it cause a right-ward shift in the H3agonist's dose-response curves. The final volume in each well is 100 μl.Test compounds are dissolved in DMSO and diluted in H₂O. The mixture isshaken for 5 min, and allowed to stand for 25 min at room temperature.The reaction is stopped with 100 μl “Detection Mix” per well. The platesare then sealed with plastic, shaken for 30 min, allowed to standovernight, and finally the radioactivity is counted in the Cobra II autogamma topcounter. EC₅₀ values are calculated by non-linear regressionanalysis of dose response curves (6 points minimum) using GraphPadPrism. Kb values are calculated by Schild plot analysis.

Functional Assay II

The ability of the compounds to bind and interact with the human, monkeyor rat H3 receptor as agonists, inverse agonists and/or antagonists, isdetermined by a functional assay, named [³⁵S] GTPγS assay.

The human H3 receptor has the following sequence:Met-Glu-Arg-Ala-Pro-Pro-Asp-Gly-Pro-Leu-Asn-Ala-Ser-Gly-Ala-Leu-Ala-Gly-Glu-Ala-Ala-Ala-Ala-Gly-Gly-Ala-Arg-Gly-Phe-Ser-Ala-Ala-Trp-Thr-Ala-Val-Leu-Ala-Ala-Leu-Met-Ala-Leu-Leu-Ile-Val-Ala-Thr-Val-Leu-Gly-Asn-Ala-Leu-Val-Met-Leu-Ala-Phe-Val-Ala-Asp-Ser-Ser-Leu-Arg-Thr-Gln-Asn-Asn-Phe-Phe-Leu-Leu-Asn-Leu-Ala-Ile-Ser-Asp-Phe-Leu-Val-Gly-Ala-Phe-Cys-Ile-Pro-Leu-Tyr-Val-Pro-Tyr-Val-Leu-Thr-Gly-Arg-Trp-Thr-Phe-Gly-Arg-Gly-Leu-Cys-Lys-Leu-Trp-Leu-Val-Val-Asp-Tyr-Leu-Leu-Cys-Thr-Ser-Ser-Ala-Phe-Asn-Ile-Val-Leu-Ile-Ser-Tyr-Asp-Arg-Phe-Leu-Ser-Val-Thr-Arg-Ala-Val-Ser-Tyr-Arg-Ala-Gln-Gln-Gly-Asp-Thr-Arg-Arg-Ala-Val-Arg-Lys-Met-Leu-Leu-Val-Trp-Val-Leu-Ala-Phe-Leu-Leu-Tyr-Gly-Pro-Ala-Ile-Leu-Ser-Trp-Glu-Tyr-Leu-Ser-Gly-Gly-Ser-Ser-Ile-Pro-Glu-Gly-His-Cys-Tyr-Ala-Glu-Phe-Phe-Tyr-Asn-Trp-Tyr-Phe-Leu-Ile-Thr-Ala-Ser-Thr-Leu-Glu-Phe-Phe-Thr-Pro-Phe-Leu-Ser-Val-Thr-Phe-Phe-Asn-Leu-Ser-Ile-Tyr-Leu-Asn-Ile-Gln-Arg-Arg-Thr-Arg-Leu-Arg-Leu-Asp-Gly-Ala-Arg-Glu-Ala-Ala-Gly-Pro-Glu-Pro-Pro-Pro-Glu-Ala-Gln-Pro-Ser-Pro-Pro-Pro-Pro-Pro-Gly-Cys-Trp-Gly-Cys-Trp-Gln-Lys-Gly-His-Gly-GiU-Ala-Met-Pro-Leu-His-Arg-Tyr-Gly-Val-Gly-Glu-Ala-Ala-Val-Gly-Ala-G1u-Ala-Gly-Glu-Ala-Thr-Leu-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser-Val-Ala-Ser-Pro-Thr-Ser-Ser-Ser-Gly-Ser-Ser-Ser-Arg-Gly-Thr-Glu-Arg-Pro-Arg-Ser-Leu-Lys-Arg-Gly-Ser-Lys-Pro-Ser-Ala-Ser-Ser-Ala-Ser-Leu-Glu-Lys-Arg-Met-Lys-Met-Val-Ser-Gln-Ser-Phe-Thr-Gln-Arg-Phe-Arg-Leu-Ser-Arg-Asp-Arg-Lys-Val-Ala-Lys-Ser-Leu-Ala-Val-Ile-Val-Ser-Ile-Phe-Gly-Leu-Cys-Trp-Ala-Pro-Tyr-Thr-Leu-Leu-Met-Ile-Ile-Arg-Ala-Ala-Cys-His-Gly-His-Cys-Val-Pro-Asp-Tyr-Trp-Tyr-Glu-Thr-Ser-Phe-Trp-Leu-Leu-Trp-Ala-Asn-Ser-Ala-Val-Asn-Pro-Val-Leu-Tyr-Pro-Leu-Cys-His-His-Ser-Phe-Arg-Arg-Ala-Phe-Thr-Lys-Leu-Leu-Cys-Pro-Gln-Lys-Leu-Lys-Ile-Gln-Pro-His-Ser-Ser-Leu-Glu-His-Cys-Trp-Lys

The monkey H3 receptor has the following sequence:Met-Glu-Arg-Ala-Pro-Pro-Asp-Gly-Pro-Leu-Asn-Ala-Ser-Gly-Ala-Leu-Ala-Gly-Glu-Ala-Ala-Ala-Ala-Gly-Gly-Ala-Arg-Gly-Phe-Ser-Ala-Ala-Trp-Thr-Ala-Val-Leu-Ala-Ala-Leu-Met-Ala-Leu-Leu-Ile-Val-Ala-Thr-Val-Leu-Gly-Asn-Ala-Leu-Val-Met-Leu-Ala-Phe-Val-Ala-Asp-Ser-Ser-Leu-Arg-Thr-Gln-Asn-Asn-Phe-Phe-Leu-Leu-Asn-Leu-Ala-Ile-Ser-Asp-Phe-Leu-Val-Gly-Ala-Phe-Cys-Ile-Pro-Leu-Tyr-Val-Pro-Tyr-Val-Leu-Thr-Gly-Arg-Trp-Thr-Phe-Gly-Arg-Gly-Leu-Cys-Lys-Leu-Trp-Leu-Val-Val-Asp-Tyr-Leu-Leu-Cys-Thr-Ser-Ser-Ala-Phe-Asn-Ile-Val-Leu-Ile-Ser-Tyr-Asp-Arg-Phe-Leu-Ser-Val-Thr-Arg-Ala-Val-Ser-Tyr-Arg-Ala-Gln-Gln-Gly-Asn-Thr-Arg-Arg-Ala-Val-Arg-Lys-Met-Leu-Leu-Val-Trp-Val-Leu-Ala-Phe-Leu-Leu-Tyr-Gly-Pro-Ala-Ile-Leu-Ser-Trp-Glu-Tyr-Leu-Ser-Gly-Gly-Ser-Ser-Ile-Pro-Glu-Gly-His-Cys-Tyr-Ala-Glu-Phe-Phe-Tyr-Asn-Trp-Tyr-Phe-Leu-Ile-Thr-Ala-Ser-Thr-Leu-Glu-Phe-Phe-Thr-Pro-Phe-Leu-Ser-Val-Thr-Phe-Phe-Asn-Leu-Ser-Ile-Tyr-Leu-Asn-Ile-Gln-Arg-Arg-Thr-Arg-Leu-Arg-Leu-Asp-Gly-Ala-Arg-G1u-Ala-Gly-Gly-Pro-Glu-Pro-Pro-Pro-Glu-Ala-Gln-Pro-Ser-Pro-Pro-Pro-Pro-Pro-Gly-Cys-Trp-Gly-Cys-Trp-Gln-Lys-Gly-His-Gly-Glu-Ala-Met-Pro-Leu-His-Arg-Tyr-Gly-Val-Gly-Glu-Ala-Ala-Ala-Gly-Ala-Glu-Ala-Gly-Glu-Thr-Ala-Leu-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser-Ala-Ala-Ser-Pro-Thr-Ser-Ser-Ser-Gly-Ser-Ser-Ser-Arg-Gly-Thr-Glu-Arg-Pro-Arg-Ser-Leu-Lys-Arg-Gly-Ser-Lys-Pro-Ser-Ala-Ser-Ser-Ala-Ser-Leu-G1u-Lys-Arg-Met-Lys-Met-Val-Ser-Gln-Ser-Phe-Thr-Gln-Arg-Phe-Arg-Leu-Ser-Arg-Asp-Arg-Lys-Val-Ala-Lys-Ser-Leu-Ala-Val-Ile-Val-Ser-Ile-Phe-Gly-Leu-Cys-Trp-Ala-Pro-Tyr-Thr-Leu-Leu-Met-Ile-Ile-Arg-Ala-Ala-Cys-His-Gly-His-Cys-Val-Pro-Asp-Tyr-Trp-Tyr-Glu-Thr-Ser-Phe-Trp-Leu-Leu-Trp-Ala-Asn-Ser-Ala-Val-Asn-Pro-Val-Leu-Tyr-Pro-Leu-Cys-His-His-Ser-Phe-Arg-Arg-Ala-Phe-Thr-Lys-Leu-Leu-Cys-Pro-Gln-Lys-Leu-Lys-Ile-Gln-Pro-His-Ser-Ser-Leu-Glu-Gln-Cys-Trp-Lys

The rat H3 receptor has the following sequence:Met-Glu-Arg-Ala-Pro-Pro-Asp-Gly-Leu-Met-Asn-Ala-Ser-Gly-Thr-Leu-Ala-Gly-Glu-Ala-Ala-Ala-Ala-Gly-Gly-Ala-Arg-Gly-Phe-Ser-Ala-Ala-Trp-Thr-Ala-Val-Leu-Ala-Ala-Leu-Met-Ala-Leu-Leu-Ile-Val-Ala-Thr-Val-Leu-Gly-Asn-Ala-Leu-Val-Met-Leu-Ala-Phe-Val-Ala-Asp-Ser-Ser-Leu-Arg-Thr-Gln-Asn-Asn-Phe-Phe-Leu-Leu-Asn-Leu-Ala-Ile-Ser-Asp-Phe-Leu-Val-Gly-Ala-Phe-Cys-Ile-Pro-Leu-Tyr-Val-Pro-Tyr-Val-Leu-Thr-Gly-Arg-Trp-Thr-Phe-Gly-Arg-Gly-Leu-Cys-Lys-Leu-Trp-Leu-Val-Val-Asp-Tyr-Leu-Leu-Cys-Ala-Ser-Ser-Val-Phe-Asn-Ile-Val-Leu-Ile-Ser-Tyr-Asp-Arg-Phe-Leu-Ser-Val-Thr-Arg-Ala-Val-Ser-Tyr-Arg-Ala-Gln-Gln-Gly-Asp-Thr-Arg-Arg-Ala-Val-Arg-Lys-Met-Ala-Leu-Val-Trp-Val-Leu-Ala-Phe-Leu-Leu-Tyr-Gly-Pro-Ala-Ile-Leu-Ser-Trp-Glu-Tyr-Leu-Ser-Gly-Gly-Ser-Ser-Ile-Pro-Glu-Gly-His-Cys-Tyr-Ala-Glu-Phe-Phe-Tyr-Asn-Trp-Tyr-Phe-Leu-Ile-Thr-Ala-Ser-Thr-Leu-Glu-Phe-Phe-Thr-Pro-Phe-Leu-Ser-Val-Thr-Phe-Phe-Asn-Leu-Ser-Ile-Tyr-Leu-Asn-Ile-Gln-Arg-Arg-Thr-Arg-Leu-Arg-Leu-Asp-Gly-Gly-Arg-Glu-Ala-Gly-Pro-Glu-Pro-Pro-Pro-Asp-Ala-Gln-Pro-Ser-Pro-Pro-Pro-Ala-Pro-Pro-Ser-Cys-Trp-Gly-Cys-Trp-Pro-Lys-Gly-His-Gly-Glu-Ala-Met-Pro-Leu-His-Arg-Tyr-Gly-Val-Gly-Glu-Ala-Gly-Pro-Gly-Val-Glu-Ala-Gly-Glu-Ala-Ala-Leu-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ala-Ala-Ala-Ser-Pro-Thr-Ser-Ser-Ser-Gly-Ser-Ser-Ser-Arg-Gly-Thr-Glu-Arg-Pro-Arg-Ser-Leu-Lys-Arg-Gly-Ser-Lys-Pro-Ser-Ala-Ser-Ser-Ala-Ser-Leu-Glu-Lys-Arg-Met-Lys-Met-Val-Ser-Gln-Ser-Ile-Thr-Gln-Arg-Phe-Arg-Leu-Ser-Arg-Asp-Lys-Lys-Val-Ala-Lys-Ser-Leu-Ala-Ile-Ile-Val-Ser-Ile-Phe-Gly-Leu-Cys-Trp-Ala-Pro-Tyr-Thr-Leu-Leu-Met-Ile-Ile-Arg-Ala-Ala-Cys-His-Gly-Arg-Cys-Ile-Pro-Asp-Tyr-Trp-Tyr-Glu-Thr-Ser-Phe-Trp-Leu-Leu-Trp-Ala-Asn-Ser-Ala-Val-Asn-Pro-Val-Leu-Tyr-Pro-Leu-Cys-His-Tyr-Ser-Phe-Arg-Arg-Ala-Phe-Thr-Lys-Leu-Leu-Cys-Pro-Gln-Lys-Leu-Lys-Val-Gln-Pro-His-Gly-Ser-Leu-Glu-Gln-Cys-Trp-Lys

The assay measures the activation of G proteins by catalyzing theexchange of guanosine 5′-diphosphate (GDP) by guanosine 5′-triphosphate(GTP) at the α-subunit. The GTP-bounded G proteins dissociate into twosubunits, Gα_(GTP) and Gαγ, which in turn regulate intracellular enzymesand ion channels. GTP is rapidly hydrolysed by the Gα-subunit (GTPases)and the G protein is deactivated and ready for a new GTP exchange cycle.To study the function of ligand induced G protein coupled receptor(GPCR) activation by an increase in guanine nucleotide exchange at the Gproteins, the binding of [³⁵S]-guanosine-5′-O-(3-thio) triphosphate([³⁵S] GTPγS), a non-hydrolysed analogue of GTP, is determined. Thisprocess can be monitored in vitro by incubating cell membranescontaining the G protein coupled receptor H3 with GDP and [³⁵S] GTPγS.Cell membranes are obtained from CHO cells stably expressing the humanH3 receptor or from HEK 293 cells stably expressing the rat or monkey H3receptor. The cells are washed twice in PBS, harvested with PBS+1 mMEDTA, pH 7.4 and centrifuged at 280 g for 5 min. The cell pellet ishomogenized in 10 ml ice-cold Hepes buffer (20 mM Hepes, 10 mM EDTA pH7.4 (NaOH)) using an Ultra-Turrax homogenizer for 30 seconds andcentrifuged for 15 min at 30.000 g. Following this centrifugation step,the membrane pellet is resuspended in 10 ml ice-cold Hepes buffer (20 mMHepes, 0.1 mM EDTA pH 7.4 (NaOH)) and homogenized as describe above.This procedure is repeated twice except for the last homogenizationstep, the protein concentration is determined and membranes are dilutedto a protein concentration of 2 mg/ml, aliquoted and kept at −80° C.until use.

In order to study the presence and the potency of an inverseagonist/antagonist the H3-receptor agonist ligand R-α-methyl histamine(RAMHA) is added. The ability of the test compound to counteract theeffect of RAMHA is measured. When studying the effect of an agonistRAMHA is not added to the assay medium. The test compound is diluted inthe assay buffer (20 mM HEPES, 120 mM NaCl, 10 mM MgCl₂ pH 7.4 (NaOH))at various concentrations followed by addition of 10⁻⁸ nM RAMHA (only inthe case where an inverse agonist/antagonist is examined), 3 μM GDP, 2.5μg membranes, 0.5 mg SPA beads and 0.1 nM [³⁵S] GTPγS and incubated for2 hours by slightly shaking at room temperature. For the rat and monkeyH3 receptor 10 μg membranes including 10 μg/ml saponin are used. Theplates are centrifuged at 420 g for 10 min and the radioactivity ismeasured using a Top-counter. The results are analyzed by non linearregression and the IC₅₀ value is determined.

RAMHA and other H3 agonists stimulate the binding of [³⁵S] GTPγS tomembranes expressing the H3 receptor. In the antagonist/inverse agonisttest, the ability of increasing amounts of test compound to inhibit theincreased [³⁵S] GTPγS binding by 10⁻⁸ M RAMHA is measured as a decreasein radioactivity signal. The IC₅₀ value determined for an antagonist isthe ability of this compound to inhibit the effect of 10⁻⁸ M RAMHA by50%. In the agonist test, the ability of increasing amounts of testcompound is measured as an increase in radioactivity signal. The EC₅₀value determined for an agonist, is the ability of this compound toincrease the signal by 50% of the maximal signal that is obtained by 10⁵M RAMHA.

Preferably, the antagonists and agonists according to the invention havean IC₅₀/EC₅₀ value as determined by one or more of the assays of lessthan 10 μM, more preferred of less than 1 μM, and even more preferred ofless than 500 nM, such as of less than 100 nM.

The Open Cage Schedule-Fed Rat Model

The ability of the present compounds to reduce weight is determinedusing the in vivo open cage Schedule-fed rat model.

Sprague-Dawley (SD) male rats of an age of about 1½ to 2 months and aweight of about 200-250 g are purchased from Mølleg{dot over (a)}rdBreeding and Research Centre A/S (Denmark). On arrival they are allowedsome days of acclimatisation before being placed in individual openplastic cages. They are habituated to the presence of food (Altrominpelleted rat chow) in their home cage only during 7 hours in the morningfrom 07.30 to 14.30 all days a week. Water is present ad libitum. As theconsumption of food has stabilised after 7 to 9 days, the animals areready for use.

Each animal is used only once to avoid carry-over effects betweentreatments. During the test sessions, the test compound is administeredintraperitoneally or orally 30 min before the start of the sessions. Onegroup of animals is administered the test compound at different dosesand a control group of animals is given a vehicle. Food and water intakeare monitored at 1, 2 and 3 hours post administration.

Any side effects may rapidly be discovered (barrel-rolling, bushy furetc.) since the animals are kept in transparent plastic cages to enablecontinuous monitoring.

1. A compound selected from the following group:1-(3,4-Dimethoxyphenyl)-4-((S)-hexahydropyrrolo[1,2-a]pyrazin-2-yl)butane-1,4-dione,1-(4-Chlorophenyl)-4-((S)-hexahydropyrrolo[1,2-a]pyrazin-2-yl)butane-1,4-dione,1-(4-Chlorophenyl)-4-(octahydropyrido[1,2-a]pyrazin-2-yl)butane-1,4-dione,1-(3-Fluoro-4-methoxyphenyl)-4-(octahydropyrido[1,2-a]pyrazin-2-yl)butane-1,4-dione,1-(3,4-Dimethoxyphenyl)-4-(octahydropyrido[1,2-a]pyrazin-2-yl)butane-1,4-dione,1-(3-Fluoro-4-methoxyphenyl)-4-((S)-octahydropyrido[1,2-a]pyrazin-2-yl)butane-1,4-dione,1-(3-Fluoro-4-methoxyphenyl)-4-((R)-octahydropyrido[1,2-a]pyrazin-2-yl)butane-1,4-dione,(S)-Octahydropyrido[1,2-a]pyrazine-2-carboxylic acid2-(4-methoxyphenyl)ethyl ester,(R)-Octahydropyrido[1,2-a]pyrazine-2-carboxylic acid2-(3-(trifluoromethyl)phenyl)ethyl ester,[3,5-Difluoro-4-((R)-octahydropyrido[1,2-a]pyrazin-2-yl)phenyl]-[phenyl]methanone,(4-Fluorophenyl)-[4-((R)-octahydropyrido[1,2-a]pyrazin-2-yl)phenyl]methanone,{[4-((R)-Octahydropyrido[1,2-a]pyrazin-2-yl)phenyl]}-{phenyl}methanone,and(R)-2-[6-(4-Trifluoromethyl-phenyl)-pyridazin-3-yl]-octahydro-pyrido[1,2-a]pyrazine,as well as any diastereomer or enantiomer or tautomeric form thereof ormixtures of these or a pharmaceutically acceptable salt thereof.
 2. Apharmaceutical composition comprising, as an active ingredient, at leastone compound according to claim 1 together with one or morepharmaceutically acceptable carriers or excipients.
 3. A pharmaceuticalcomposition according to claim 2 in unit dosage form, comprising fromabout 0.05 mg to about 1000 mg of the compound.
 4. A pharmaceuticalcomposition according to claim 2 in unit dosage form, comprising fromabout 0.1 mg to about 500 mg of the compound.
 5. A pharmaceuticalcomposition according to claim 2 in unit dosage form, comprising fromabout 0.5 mg to about 200 mg of the compound.